Tolerance to heated egg in egg allergy: Explanations and implications for prevention and treatment

Abstract Hen's egg allergy is the second most frequent food allergy found in children. Allergic symptoms can be caused by raw or heated egg, but a majority of egg‐allergic children can tolerate hard‐boiled or baked egg. Understanding the reasons for the tolerance towards heated egg provides clues about the molecular mechanisms involved in egg allergy, and the differential allergenicity of heated and baked egg might be exploited to prevent or treat egg allergy. In this review, we therefore discuss (i) why some patients are able to tolerate heated egg; by highlighting the structural changes of egg white (EW) proteins upon heating and their impact on immunoreactivity, as well as patient characteristics, and (ii) to what extent heated or baked EW might be useful for primary prevention strategies or oral immunotherapy. We describe that the level of immunoreactivity towards EW helps to discriminate patients tolerant or reactive to heated or baked egg. Furthermore, the use of heated or baked egg seems effective in primary prevention strategies and might limit adverse reactions. Oral immunotherapy is a promising treatment strategy, but it can sometimes cause significant adverse events. The use of heated or baked egg might limit these, but current literature is insufficient to conclude about its efficacy.

children are IgE-mediated reactions, such as erythema, urticaria, eczematous rash, abdominal pain, diarrhoea and vomiting. 6The current treatment for egg allergy involves strict dietary avoidance or minimised contact with the allergen.As an alternative to an avoidance diet, oral immunotherapy (OIT) has been investigated.OIT involves the ingestion of small doses of egg protein by an allergic individual.This dose is gradually increased over time to improve tolerance and further desensitize the allergic patient.Beyond treatment strategies, primary prevention strategies are actively studied to prevent the development of egg allergy.These prevention strategies notably involve the early introduction of specific forms of egg in young infants.
Clinical reactivity occurs towards specific amino acid sequences of proteins, which are called epitopes.Linear epitopes are defined as continuous sequences of amino acids capable of binding IgE, whereas conformational epitopes are formed by amino acids that are spatially close in the protein 3D conformation but distant in the protein sequence.Known linear and conformational epitopes of OVA and OVM are noted in Figures 1 and 2. 37 Allergic symptoms can be caused by the consumption of raw or heated eggs.9][40] Two phenotypes of egg-allergic children can thus be distinguished; patients reactive only to raw egg and patients reactive to all forms of egg.Understanding the reasons for the tolerance of heated egg by some but not all egg-allergic patients might provide clues about the molecular mechanisms involved in hen's egg allergy sensitization and allergic reactions in general.Furthermore, the differential allergenicity of the different forms of eggs might be exploited to prevent or treat egg allergy.In this review, we aim to discuss in detail (i) why some patients are able to tolerate heated egg; by discussing the structural changes of EW proteins upon heating and their impact on EW immunoreactivity, as well as patient characteristics, and (ii) to what extent heated egg (white) might be useful for primary prevention strategies or oral immunotherapy.As OVM and OVA are the immunodominant allergens, the impact of heating on these allergens will be discussed in detail.

| WHY DO SOME PATIENTS TOLERATE HEATED EGG?
2.1 | EW heating can modify immunoreactive epitopes and protein digestion

| Structural characteristics of heated EW
To understand why heated eggs are better tolerated by egg-allergic patients, the physiochemical and structural changes occurring during the heating of EW (proteins) need to be considered first.Various types of heat treatment can be applied to egg or EW to make different food products, including egg (white) pasteurization (58-65.5°Cfor 2.5-5 min for liquid egg, 55-57.2°Cfor liquid EW), 41 boiling (100°C for 5-30 min), scrambling (pan-cooked, 4-6 min), and baking.Baked egg is characterized by a method of cooking that uses prolonged dry heat, normally in an oven, in the absence (e.g.ovenbaked egg) or the presence of wheat proteins (e.g.muffins or biscuits).With increasing temperature, EW proteins progressively unfold and denature, which results in protein aggregation and coagulation, giving heated EW its milky white colour. 42Beyond aggregation, heating can also induce the so-called Maillard glycation, which is a complex set of chemical reactions in which free amino groups of proteins interact with the carbonyls of reducing sugars. 43illard glycation takes place naturally in the presence of sugars but is accelerated by heat and is frequently observed during baking and cooking as food browning. 43The progressive unfolding of egg proteins and their glycation upon heating depends on (i) the time and temperature of heating, (ii) the characteristics of the EW proteins, and (iii) environmental factors (e.g.pH, ionic strength, the presence of other protein sources such as wheat gluten). 43 aggregation generally starts at 60°C-the denaturation temperature of ovotransferrin-and further accelerates at 70°C-the denaturation temperature of OVA. 6,44OVA contains 6 cysteine residues (Cys12, Cys31, Cys74, Cys121, Cys368, and Cys383) of which Cys74 and Cys121 form a disulphide bond in the native state.Abbreviation: EW, egg white.
Following heat denaturation, a hydrophobic C-terminal region containing a sulfhydryl group (Cys368) is exposed to the surface and contributes to OVA aggregate formation. 46,47OVA aggregation is rapid and results in the formation of thin strands (linear aggregates) or denser particles depending on the physicochemical conditions used during heating (pH, ionic strength, protein concentration). 12,14,48In contrast to the heat-labile nature of OVA, OVM is highly resistant to heat thanks to its conformation of three tandem domains with intra-but not inter-domain disulphide bonds. 8,44,49ly prolonged heating at temperatures above 90°C (e.g.boiling >30 min) results in the formation of an irreversible denatured state, indicating that OVM will remain in a natural state in more transiently heated forms of egg. 8,50One particular situation in which OVM does aggregate is when OVM is heated in the presence of wheat.2][53] For this reason, egg baked in the presence of wheat should be clearly distinguished from other forms of heated egg in scientific studies.

| Heating can modify immunoreactive epitopes
The heat-induced changes in EW proteins impact allergen epitopes.
Allergens have two types of epitopes; T-lymphocyte epitopes that are recognized by T-lymphocytes following protein processing and presentation by antigen-presenting cells, and B-cell or IgE epitopes.
These IgE epitopes are protein regions capable of binding and crosslinking IgE, produced by plasma cells from memory B-cells.Crosslinking of the IgE-FcεRI complex on the surface of mastocytes or basophils by an allergen causes their degranulation and the release of mediators (e.g.histamine) that provoke allergic symptoms.[34][35][36] LEAU ET AL.
epitopes can either be linear or conformational, heat treatment might destroy conformational epitopes or mask linear epitopes due to protein aggregation or glycation, which might change epitope accessibility or alternatively generate new epitopes. 54e immunoreactive epitopes in OVA have been identified as a combination of conformational and linear epitopes. 23Conformational epitopes have been localized to the regions aa41-172 (located at the surface of OVA) and aa367-385 23,24 (Figure 1).Due to the heat lability of OVA, these conformational epitopes are likely lost upon heating.
25]27 It is of interest to note that this particular epitope is also in a region that aggregates and is glycated upon heating, suggesting that this epitope might become partially masked. 46,47Other aggregation 'hotspots' for OVA have been identified at Cys31 and Cys121, which are both in proximity to linear OVA IgE epitopes (aa16-30 and aa125-134). 25,29,47'Hotspots' of glycation were found at Lys190 (within the epitope aa 189-199) for dry heated samples and at Lys123 (near epitope aa 125-134) for wet heated samples. 25,29,47,55These structural changes of OVA epitopes induced by heating and/or glycation lower the recognition of OVA epitopes by sIgE of patients, as assessed using Western blot and/or ELISA (see Table 2).Some linear epitopes do persist, as heat treatment of OVA does not fully abolish sIgE reactivity of patient sera 9 (see Table 2).Indeed, two linear epitopes (aa 229-243, 280-297) were suggested to be specific for patients sensitive to extensively heated egg. 28r OVM, both linear and conformational epitopes play a role in OVM allergy and their relative importance likely differs per patient. 63me OVM-sensitized individuals might not recognize linear epitopes at all. 32Overall, OVM heating moderately reduces serum IgE binding, but most OVM-reactive patients still react significantly to heated OVM (see Table 2).For some patients, IgE reactivity even increases upon glycation, suggesting the appearance of novel epitopes 9,10,64 (see Table 2).As OVM does not aggregate and only irreversibly denatures upon prolonged heat exposure (boiling >30 min), it is likely that many OVM IgE-binding epitopes remain accessible in moderately heated OVM and EW, although no detailed molecular studies on OVM epitopes and heating have been performed yet.Nonetheless, the reduced epitope accessibility of OVA and, to a lesser extent, OVM likely explains the reduced capacity of sIgE of egg-allergic patients to bind to heated or baked EW (see Table 2).The length of heat treatment seemed to be the most determinant for the loss of EW IgE reactivity following heating, which is probably linked to the gradual chemical modification of linear IgE epitopes 56,57 (see Table 2).

| Heating alters egg protein digestion and absorption
Beyond the changes in conformational or linear epitopes, heating also impacts the digestibility of EW proteins and their absorption.
For food allergens to trigger allergic symptoms, the allergen must conserve at least 2 epitopes following digestion and be absorbed in an immunologically active form across the epithelial barrier.An extensive study that used EW heated at different temperatures and times (56°C for 32 min; 65°C for 30 min; 100°C for 5 min) showed that heating significantly increased EW protein digestion. 16Gastric digestion was highest following heating at 65°C for 30 min, whereas gastro-intestinal digestion was highest upon heating at 100°C for 5 min. 160][11][12][13] Indeed, whereas native OVA has a high resistance to gastric digestion, heat-aggregated OVA is more easily digested and the peptides that are released are different. 12,13The reactivity of basophils sensitized with sera from egg-allergic patients was also significantly reduced but not abolished following the heating and digestion of OVA, compared to unheated, digested OVA. 9,13 contrast to heating alone, the glycation of OVA lowers its digestibility and the peptides released are different than unheated or heated OVA. 10,65It remains to be clarified to what extent heated or glycated OVA crosses the barrier in an immunologically active form.
Two studies indicated that the heating of OVA significantly lowered the amount of circulating OVA following oral gavage in mice, while another study showed that heated OVA was unable to activate presensitized basophils following transport across the intestinal barrier. 9,66,67 contrast to OVA, OVM gastro-intestinal digestion is not significantly affected by heating due to its high thermal stability. 9,10M is digested by gastric and gastro-intestinal fluids, but its digestion is not complete as epitopes recognized by IgE in human sera remain present. 8,10,11Using basophils sensitized with sera from egg-allergic patients, gastro-intestinal degradation but not heating of OVM significantly reduced basophil reactivity. 9Glycation of OVM also did not affect gastro-intestinal digestion. 10Heating did lower OVM immunoreactivity following passage of the epithelial barrier compared with native OVM, but the underlying mechanisms remain to be clarified. 9

Summary:
� OVA is more heat labile than OVM -5 of 24

| Heating egg lowers allergic sensitization capacity
To explain why certain patients tolerate heated or baked egg, multiple studies have studied how heating impacts the capacity of EW (proteins) to sensitize or provoke an allergic reaction.For allergic sensitization, data on the sensitization capacity of raw versus heated egg are only available for mice studies and are largely inconclusive.In one study, a significant reduction of total IgE and OVA sIgE levels was found when mice were sensitized to heated EW, compared to raw EW. 67 In contrast, using a short heating time (10 min 80°C), another study found that mice sensitized with heated EW had significantly higher total IgE and OVA and OVM sIgE levels compared with raw EW 54 (see Table 3).
Studies using OVA to sensitize mice are more consistent and show that mice sensitized with heated OVA (10 min at 70°C or 6 h at 80°C) have modestly lower OVA sIgE compared to mice sensitized with native OVA 68,69 (see Table 3).9][70] Interestingly, the sensitization capacity of heated OVA was found to be dependent on the aggregation process: small, linear aggregates of OVA formed at pH 9 (near natural pH of stored EW) and low ionic strength displayed a reduced allergic potential compared to large, spherical agglomerated aggregates formed at pH 5 and high ionic strength. 69ly few studies have investigated the impact of glycation on the sensitization capacity of OVA.Two studies showed a reduction in serum IgE levels following the sensitization of mice with glycated OVA compared with native OVA. 67,71In contrast, a more recent study using heavily glycated OVA showed increased IgE levels and a stronger reduction in body temperature compared with intraperitoneal sensitization with native OVA. 72These opposing results are likely due to the extent of glycation and the heating temperature used to glycate OVA in the different studies and further studies are needed to clarify the impact of the extent of glycation on sensitization to OVA (Table 3).
No data on allergic sensitization of heated and/or glycated OVM versus native OVM are currently available.

| Heating egg lowers egg allergic reactions
Numerous mice studies have investigated the capacity of heated EW (protein) to elicit allergic symptoms (see Table 3).In accordance with the observations in patients, all studies demonstrated a reduction in allergic symptoms when mice are sensitized and/or elicited with heated EW (protein) 9,54,66,67,69,70 (see Table 3).Pablos-Tanarro and colleagues used an extensive cross-over design in which mice were sensitized to native or heated EW and provoked with either native or heated EW. 54 In this study, provocation with heated EW resulted in lower allergic symptoms in all mice compared to native EW, while the combined sensitization and provocation with heated EW resulted in the lowest overall clinical symptoms. 54In line with these studies, the reactivity of basophils sensitized with sera from egg-allergic patients or sensitized mice was significantly reduced upon exposure to heated OVA or OVM, when compared to the native protein (see Tables 3 and   4).No studies have, to our knowledge, investigated the elicitation capacity of glycated OVA or OVM in mice or using basophils.
When the elicitation capacity was studied in a clinical setting, several studies showed that the wheal diameter of patient skin-prick tests (SPT) using baked egg in the presence (muffin) or absence (ovenbaked) of wheat was generally smaller when compared to raw EW 73,74 (see Table 4).Similarly, using hard-boiled egg, EW or egg yolk, part of children responsive to raw egg forms were not responsive any more in SPT (43% (egg), 33% (EW) or 72% (egg yolk)). 75Pasteurization of egg or EW did not significantly affect SPT size, and only very few raw egg reactive patients became nonreactive upon pasteurization 75 (see Table 4).
In oral food challenges (OFC) that investigate the clinical reactivity profile of egg-allergic patients, a direct comparison of the reactivity towards baked/heated and uncooked eggs is generally not made (see Table 4).Instead, a patient who reacts to baked or heated egg is considered to react also to raw egg.egg) 38,[76][77][78][79] (see Table 4).Given the more complex composition of the foods tested in SPT, the relative impact of heating versus glycation is difficult to be determined in these studies.Taken together, the heating or baking of EW (proteins) significantly lowers the capacity to provoke an allergic reaction, with the most pronounced changes observed after prolonged heating.Given the heat stability of OVM, it is likely that part of the residual immunoreactivity of heated EW is due to the recognition of OVM and not OVA.In support for a role of OVM in reactions towards heated egg, a part of patients responsive to heated egg was able to consume heated eggs depleted of OVM. 76

Summary:
� OVA heating lowers its sensitization capacity, while the impact of OVM heating or OVA/OVM glycation on sensitization capacity remains to be further investigated

| Patient reactivity to heated/baked EW and patient prognostics depend on the sIgE sensitization profile
The previous sections highlight that heating has a significant impact on the sensitization capacity and allergic reactivity of EW by altering EW structure and digestion.However, to understand why certain patients react to heated egg whereas others do not, we also need to look at the patients' clinical profiles.Several studies have attempted to address this question.1][82][83] Similarly, reactivity threshold doses for children allergic to raw but not baked egg are higher than for the general population of egg allergic children. 84These observations suggest that the severity of egg allergy might be a determinant factor for being tolerant or reactive to heated eggs.However, although elevated sIgE has a predictive value for the classification of patients, no generalizable cut-offs for SPTs or sIgEs have been agreed upon so far and an OFC using heated or baked egg remains the gold standard. 82,83,85,86ven the heat stability of OVM, several studies have suggested that the sIgE levels of OVM might be used to discriminate patients responsive or tolerant to heated egg. 40,77,80,87,88However, other studies have not confirmed a predictive value of OVM sIgE levels and no cut-off for patient classification on the basis of OVM sIgE is currently available. 82,86One factor that might explain the discrepancy between studies is the usage of heated egg versus baked egg due to the aggregation of heated OVM in the presence of wheat. 51,82wever, to what extent the presence of wheat influences clinical reactivity to OVM in patients remains to be further established.A study that orally challenged egg-sensitized individuals with different food matrices suggested that the presence of wheat was only important in a minority of the patients and that the duration of egg heating (10 min vs. 30 min) was more determinant for a clinical reaction. 89Beyond the magnitude of egg sIgE levels or OVM sIgE levels, a higher reactivity to linear epitopes (that are less heataltered) in patients reactive to heated egg might also play a role. 33is type of information is, however, not obtained by measurement of sIgE binding to the entire allergen and specific epitopes that might predict the tolerance or not to heated egg would need to be confirmed. 28

| Patient prognostics
Beyond contributing to the quality of life of egg-allergic patients, a patient classification based on responsiveness to heated eggs might be useful to anticipate patient prognostics.As mentioned, many patients will outgrow hen's egg allergy, with a resolution of approximately 50% at the age of 2. 5 The ability to tolerate baked egg is predictive of the transiency of egg allergy; patients unable to tolerate baked egg are five times less likely to develop tolerance. 5In line with the characteristics distinguishing baked egg-tolerant from reactive patients, it has been proposed that patients who have higher sIgE to raw EW, that are sensitized to OVM or multiple egg allergens and that are highly reactive to linear epitopes of OVM or OVA are less likely to outgrow their egg allergy. 33,86,90,91mmary: � Patients reactive to both heated and raw eggs are characterized by higher overall sIgE levels to EW, OVA and OVM compared to patients responsive only to raw egg � Tolerance to baked egg is predictive of the transiency of egg allergy; patients unable to tolerate baked egg are five times less likely to develop tolerance

| USE OF HEATED EGG FOR PRIMARY PREVENTION AND TREATMENT OF EGG ALLERGY
also notably in their usage of different forms of eggs to achieve tolerance; for example, raw or heated egg (white).The different structure and immunological reactivity of the different forms of eggs make it of interest to assess whether and to what extent the primary prevention and treatment of egg allergy is impacted by the form in which the egg allergen is provided.

| Use of heated egg for primary prevention of egg allergy
3][94][95][96][97] Several randomized controlled trials have been conducted to evaluate the efficacy of an early introduction of egg in infants to prevent egg allergy using different types and doses of egg proteins, and different patient populations (general population, high risk) (see Table 5).In these studies, the most commonly used form of egg was pasteurized raw egg (white) powder, which has equivalent allergenic properties compared to raw egg 98 (see Table 5).Other studies used heated egg powder or boiled egg (see Table 5).A systematic review and meta-analysis 99 assessed the combined effect of the early introduction versus no early introduction of egg protein and the risk of developing an egg allergy in these randomized controlled trials.It concluded to an overall significant protective effect of early introduction of egg protein with a decreased relative risk of developing an egg allergy in the egg group versus control group of 0.60 (CI: 0.44-0.82).It is, however, important to note that a significant number of adverse reactions (31%, 94 6.1%, 93 8.1%, 92 7.1% 96 ) was described, notably in studies using pasteurized raw egg (white) powder.In contrast, the PETIT study, which used heated egg powder, did not describe any adverse events. 95The incidence of adverse reactions might also be impacted by the daily dose of egg protein given, which was high in the STAR study 94 that described a high incidence of adverse reactions, and low in the PETIT study. 95Beyond the safety profile, the efficacy might also be impacted by the type or dose of egg used, but none of the studies directly compared the use of different types of eggs in primary prevention.Nonetheless, it is clear that heated egg-with its good safety profile-is able to successfully prevent the development of egg allergy, 95,97 whereas the studies using pasteurized egg (white) powder gave more heterogeneous results (see compared with the exposition to baked eggs (OR, 0.2 [95% CI, 0.06-0.71]). 100Based on these data, it might thus be hypothesized that for the effective prevention of egg allergy, the exposition of an infant to egg epitopes should be high enough to induce tolerance but also low enough to not sensitize or provoke an allergic reaction.This balance might be modified not only by the dose of egg used but also by the form of egg protein given.Indeed, as discussed in Section 2, the heating of EW protein modifies the accessibility of linear and conformational epitopes and increases its digestibility.
The choice of the egg form to introduce into an infant's diet is of particular importance as a significant proportion of infants are already sensitized to eggs before food diversification. 94,96How these infants are sensitized to egg is not fully clear, but their sensitization might have occurred in utero through the transfer of small doses of antigen in breast milk or through a defective skin barrier (for example due to the presence of egg protein in dust). 101,102Recent studies have suggested that the exposition of infants to egg-derived allergens and egg-specific IgG in breast milk might contribute to the development of oral tolerance and a lower egg allergic risk in infants. 103,104An on-going randomized controlled trial now aims to determine whether a higher maternal egg and peanut consumption during pregnancy and lactation might prevent the development on infant egg and peanut allergy. 105

Summary:
� Heated egg might be the best form to prevent the occurrence of egg allergy, given its efficacy and safety profile.For this reason, the S3 guideline Allergy Prevention now recommends "For prevention of hen's egg allergy, well-cooked (e.g., baked or hard-boiled), but no "raw" eggs (…) should be introduced with the complementary food and given regularly.". 106

| Use of heated egg for egg allergy treatment: OIT
OIT is a potential treatment for egg allergy, consisting of the progressive reintroduction of the allergy-causing food.It includes an induction phase (IP) during which the ingested dose increases progressively to reach a target dose, and a maintenance phase (MP) during which the allergen is taken regularly.The IP often starts with an initial escalation phase with increasing doses of allergen given every 20-30 min during a day or two under clinical supervision to determine the starting dose for the IP.Patients undergoing an OIT can achieve desensitization and sometimes achieve maintained tolerance.Desensitization refers to the ability to ingest a food without reaction while continuing to take regular doses of that food, whereas maintained tolerance is the ability to tolerate a food after a period of food avoidance.The maintained tolerance is assessed by performing an oral food challenge (OFC) after discontinuing the ingestion of the allergen for a period of at least 4 weeks.
Many studies have investigated the effectiveness of OIT in egg allergy, including randomized controlled trials, uncontrolled trials, and observational studies.We will focus here on 15 randomized controlled trials (see Table 6).Although many of these studies included only a few patients, the data provided by these studies indicate that the efficacy of egg allergy OIT is generally very good, although mild-to-moderate adverse events are very frequent (see Table 6).This observation was confirmed by a meta-analysis that included 10 randomized controlled trials and concluded to the efficacy of OIT compared with a control group: most children (82%) in the OIT group could ingest a partial serving of raw or undercooked egg (1-7.5 g) compared to 10% of control group children. 123It should be noted, however, that in the different studies the inclusion criteria, dosage, target dose, and the duration of the IP and MP are diverse (see Table 6).Especially dosing and frequency of exposition seem quite important for tolerance induction, as demonstrated in the SEICAP study that compared two protocols of OIT. 115In this study, one group increased their daily egg intake with 5% and their weekly intake with 30%, whereas a second group had only a 30% weekly updosing; the first pattern was more effective than the second. 115fferent types of eggs were used in the different OIT trials (see Table 6).In general, most studies used a rather 'native' form of egg (white) for OIT trials, such as dehydrated egg, pasteurized egg (white) powder or liquid, or raw hen's egg emulsion.Dehydrated egg powder was most commonly tested and generally compared to a control group having either a placebo or an egg avoidance diet.Although different protocols were used, in all of these studies OIT was associated with an increased percentage of desensitization and maintained tolerance compared with the control group (see Table 6).One study that did not show efficacy used a low-allergenic hydrolysed form of egg, but this study also did not use dose increments. 122Two randomized controlled trials specifically assessed the efficacy of baked egg consumption to induce oral tolerance in egg-allergic patients, 116,119 as did one non-randomized clinical trial. 124Indeed, earlier studies suggested that the regular ingestion of baked egg in egg allergic children could accelerate the development of egg tolerance. 5,125In a small, non-randomized clinical trial, the incremental ingestion of baked egg (from 125 mg to 3.8 g of baked egg daily) was shown to induce progressive desensitization to baked egg and lightly cooked egg (cooking conditions not specified). 124Importantly, compared to other OITs, only very few adverse events were reported. 124In contrast, in a randomized clinical trial that included a control group of egg-avoiding patients, the regular ingestion of the same dose of 10 g of baked egg (equivalent to 1.3 g egg protein) for 6 months did not increase the proportion of patients who were able to pass an OFC to raw egg 1 month after ceasing the intervention. 119 significant differences in adverse events were reported between the baked egg-consuming group and the control group. 119 to 2 g EW protein daily, no dose increments) and compared this protocol to an OIT using pasteurized EW powder in baked eggtolerant patients (up dosing to 2 g pasteurized EW protein). 116In this study, regular baked egg consumption was less effective to induce sustained unresponsiveness than the OIT approach with pasteurized EW powder, with an equivalent safety profile. 116No randomized clinical studies have directly compared an OIT using baked or heated eggs with an OIT using a raw or pasteurized form of egg, although a randomized non-controlled study suggested that heated eggs can be effectively used in OIT. 126Given the lower allergenicity of heated or baked egg, it might be hypothesized that the usage of baked or heated egg might provide a more favourable safety profile, especially in the initial steps of OIT.In some countries, so-called food ladders are now tools used to progressively reintroduce common foods containing eggs into the diet of egg-allergic children and to induce tolerance.These food ladders consist of a step-wise gradual introduction of increasingly allergenic forms of egg at home, starting from extensively heated to less heated eggs.These food ladders could be considered as a form of OIT, but they still lack standardization and a sound scientific underpinning of their efficacy. 127,128mmary: � OIT is an effective approach to promote desensitization and maintain tolerance in egg-allergic patients � Dehydrated egg powder is the most commonly tested form of egg in OIT � The usage of heated or baked forms of egg might be an option for OIT, but more research is needed to confirm preliminary studies

| CONCLUSION
To understand and establish strategies for the diagnostics, treatment and prevention of food allergy, detailed information about the responsible allergens is required.In the case of hen's egg allergy, a part of the patients is reactive to raw but not extensively heated or baked egg.The reasons for this seem to be F I G U R E 3 Overview of the physicochemical characteristics of egg white (EW) proteins and the patient characteristics that are potential determinants for the tolerance of patients towards heated eggs.The impact of egg heating on primary prevention strategies and oral immunotherapy is also noted.
LEAU ET AL.
multiple and relate to the physiochemical properties of the heated egg allergens on the one hand, and patient reactivity on the other hand (see Figure 3).Heating notably impacts the protein conformation and digestibility of the major EW protein OVA, whereas heating only impacts OVM upon prolonged heating or when wheat is present.On the patient side, the overall immunoreactivity towards hen's EW appears to be determinant for the discrimination of patient tolerant or reactive to heated or baked egg.Other implicated factors are patient reactivity to the heatstable OVM and to linear versus conformational epitopes, but these factors require further experimental validation.For primary prevention strategies of egg allergy, the use of a heated/baked form of egg might limit adverse reactions when compared to pasteurized raw egg powder and effectively prevent the egg allergy.A lightly heated or baked form of egg might also be an interesting option, in order to ensure that an individual is sufficiently exposed to egg epitopes to induce tolerance, but that the risk of sensitizing or provoke an allergic reaction is low.OIT seems to be a promising treatment for egg allergy, but significant adverse events have been reported.The use of heated or baked egg could be an interesting option to limit these adverse events, but the current literature is insufficient to conclude the efficacity of such an approach.Taken together, a good understanding of the impact of food transformation on its allergenicity might be helpful to ameliorate primary prevention and treatment strategies for food allergies.

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LEAU ET AL.T A B L E 4 IgE patient serum reactivity against native and heated EW proteins.= dependent on patient, compared to EW 74 EW, egg yolk, or whole egg 200°C for 25 min EW, egg yolk, or whole egg baked in an oven ↓ compared to uncooked egg 36 children with egg allergy 73 EW, egg yolk, or whole egg 94°C for 18 min Hardboiled egg, EW, or egg yolk ↓↓ for 43% (egg), 33% (EW), 72% (egg yolk) of children, = for the rest of children for egg and EW, Heating of EW (proteins) lowers its capacity to induce an allergic reaction in mice � Heated or baked egg white (proteins) has a lower sIgE binding capacity and lower SPT wheal diameter compared to raw egg � A significant proportion of egg-allergic patients irresponsive to heated/baked egg (white) react to raw egg in OFC � The impact of heating or baking on allergic reactivity is dependent on the time and temperature of heating Leau: Conceptualization (supporting); formal analysis (equal); investigation (equal); writing-original draft (supporting).Sandra Denery-Papini: Conceptualization (supporting); supervision (supporting); validation (supporting); writing-review and editing (supporting).Marie Bodinier: Conceptualization (supporting); supervision (supporting); validation (supporting); writing-review and editing (supporting).Wieneke Dijk: Conceptualization (lead); formal analysis (equal); funding acquisition (lead); investigation (equal); resources (lead); supervision (lead); visualization (lead); writing-original draft (lead). 45 Major allergens in hen's eggs.
T A B L E 1

4 of 24 - LEAU ET AL. T A B L E 2 IgE patient serum reactivity against native and heated EW proteins. Protein fraction Heating Heating conditions Product Western blot/dot blot ELISA References
LEAU ET AL.

Table 5
).In line with this, an observational study noted that exposure to cooked egg (defined as boiled, scrambled, fried, or poached) but not to baked egg (defined as egg-containing cakes or biscuits or similar products) induced the development of oral tolerance: at 4-6 months of age, the first exposition to cooked eggs reduced the risk of egg allergy T A B L E 5 Randomized controlled primary intervention trials for egg allergy.-LEAUET AL.
Randomized controlled OIT trials for egg allergy.BE, baked egg; EW, egg white; IP, induction phase; MP, maintenance phase; N/A, not assessed or not reported; OFC, oral food challenge.
T A B L E 6 (Continued) -LEAU ET AL.